Detection of Potato mop top virus and Tobacco rattle virus using a multiplex real-time fluorescent reverse-transcription polymerase chain reaction assay
Ra. Mumford et al., Detection of Potato mop top virus and Tobacco rattle virus using a multiplex real-time fluorescent reverse-transcription polymerase chain reaction assay, PHYTOPATHOL, 90(5), 2000, pp. 448-453
Tobacco rattle virus (TRV) and Potato mop top virus (PMTV) are important di
seases of potato that are difficult to diagnose reliably by visual symptoms
. Effective control strategies rely on accurate diagnosis. This paper descr
ibes the development of a multiplex assay for the detection of TRV and PMTV
directly from potato tubers and leaves by polymerase chain reaction (PCR)
combined with in-tube fluorescent product detection (TaqMan). This technolo
gy obviates any post-PCR manipulations and has many advantages including re
ducing contamination risks, eliminating the need for ethidium bromide stain
ing, and removing the time and cost of gel running. The new assay also allo
ws the replacement of the two separate tests (a TRV reverse-transcription-P
CR and a PMTV enzyme-linked immunosorbent assay) currently used with a sing
le-tube multiplex format. In addition to greatly simplifying the detection
of these two viruses, the multiplex TaqMan assay was also shown to be more
sensitive than either of the tests that it replaces, allowing 100- and 10,0
00-fold increases in sensitivity for TRV and PMTV detection, respectively.
The test reliably detected over 40 different isolates of TRV and PMTV obtai
ned from a wide range of different cultivars and geographical locations, in
cluding some samples in which existing tests failed to detect virus. The us
e of an assay of this kind in routine diagnosis helps to speed up and strea
mline the diagnostic laboratory; in addition, more reliable diagnosis shoul
d help in the control of this damaging disease.